The present invention relates to multipotent neural stem cells (MNSCs) purified from peripheral tissues containing sensory receptors such as the skin, olfactory epithelium, and tongue.
There are a number of diseases of the central nervous system (xe2x80x9cCNSxe2x80x9d) which have a devastating effect on patients. These diseases are debilitating, often incurable, and include, for example, Alzheimer""s disease, Huntington""s disease, Parkinson""s disease, and Multiple Sclerosis.
By way of example, Parkinson""s disease is a progressive degenerative disorder of unknown cause. In healthy brain tissue, dopaminergic neurons extend from the substantia nigra of the brain into the neighboring striatum. In Parkinson""s disease, these dopaminergic neurons die.
There are a number of methods to treat Parkinson""s disease. One method is to treat humans having Parkinson""s disease with L-DOPA. A second method is to transplant cells into the substantia nigra or striatum. Transplanted cells replace endogenous cells that are lost as a consequence of disease progression. An animal model of Parkinson""s disease is an MPTP-treated non-human primate. The MPTP-treated animals have been transplanted with dopamine-rich embryonic neurons with some success.
To date, the cells used for neural transplant have been collected from the developing brains of aborted fetuses. Aside from the ethical considerations, the method from a practical standpoint is unlikely to provide a sufficient amount of neural tissue to meet the demands. Thus, another source of cells for transplantation is desirable.
Stem cells are undifferentiated cells that exist in many tissues of embryos and adult mammals. In embryos, blastocyst stem cells are the source of cells which differentiate to form the specialized tissues and organs of the developing fetus. In adults, specialized stem cells in individual tissues are the source of new cells, replacing cells lost through cell death due to natural attrition, disease, or injury. Stem cells may be used as substrates for producing healthy tissue where a disease, disorder, or abnormal physical state has destroyed or damaged normal tissue.
MNSCs may be used as a source of cells for transplantation. The MNSCs may themselves be transplanted or, alternatively, they may be induced to produce differentiated cells (e.g., neurons, oligodendrocytes, Schwann cells, or astrocytes) for transplantation. Transplanted stem cells may also be used as vectors for the expression of therapeutic molecules, such as growth factors, cytokines, anti-apoptotic proteins, and the like. Thus, stem cells are a potential source of cells for alternative treatments of diseases involving loss of cells or tissues.
The safest type of tissue graft (using stem cells or otherwise) is one that comes from self (an autologous tissue source). Autologous tissue sources are widely used in procedures such as bone transplants and skin transplants because a source of healthy tissue is readily accessible for transplant to a damaged tissue site. In brain diseases, such as Parkinson""s disease, healthy dopaminergic neuronal brain tissue may exist at other sites in the brain, but attempts to transplant these neurons may harm the site where the healthy neurons originate. MNSCs that can be differentiated into dopaminergic neurons may be available at other sites from which they may be transplanted, but the CNS, particularly the brain, is physically difficult to access.
In several tissues, stem cells have been purified and characterized. For example, MNSCs have been purified from the mammalian forebrain (Reynolds and Weiss, Science 255:1707-1710, 1992) and these cells were shown to be capable of differentiating into neurons, astrocytes, and oligodendrocytes. PCT publications WO 93/01275, WO 94/16718, WO 94/10292 and WO 94/09119 describe uses for these cells. It could be impractical or impossible, however, to first access brain or other CNS tissue for biopsy and then again for transplant in patients with weakened health. It would be very useful if there were accessible stem cells capable of differentiating into CNS cell types, such as dopaminergic neurons; such cells would be a source of cells for autologous transplants.
Thus, there is a clear need to develop methods for identifying from accessible tissues neural stem cells that can act as a source of cells that are transplantable to the CNS, PNS, or other tissues in vivo in order to replace damaged or diseased tissue.
We have substantially purified MNSCs from the peripheral tissue of postnatal mammals, including juvenile and adult mammals. Most importantly, we have identified skin as a source of MNSCs and provide methods for the purification of skin-derived MNSCs, thus simplifying the harvesting of cells for transplantation relative to previous methods. The MNSCs possess desirable features in that they are multipotent and self-renewing. The cells can be repeatedly passaged and differentiated into cell types of the CNS, including astrocytes, oligodendrocytes, and neurons. The MNSCs express nestin, an immunological marker of neural stem cells and progenitor cells. The cells are capable of differentiating as dopaminergic neurons, and thus are useful source of dopaminergic neurons for homotypic grafts into Parkinson""s Disease patients. The MNSCs may also make non-neural cells such as cardiac muscle cells, pancreatic islet cells, smooth muscle cells, hematopoietic cells, adipocytes, hepatocytes, and the like. The cells may also be used for autologous or heterologous transplants to treat, for example, other neurodegenerative diseases, disorders, or abnormal physical states.
Accordingly, in a first aspect, the invention features a MNSC substantially purified from a peripheral tissue of a postnatal mammal, wherein the peripheral tissue includes a sensory receptor.
In a second aspect, the invention features a cell that is the progeny of a MNSC substantially purified from a peripheral tissue of a postnatal mammal. The cell may be a mitotic cell or a differentiated cell (e.g., a neuron, an astrocyte, an oligodendrocyte, a Schwann cell, or a non-neural cell). Preferred neurons include neurons expressing one or more of the following neurotransmitters: dopamine, GABA, glycine, acetylcholine, glutamate, and serotonin. Preferred non-neural cells include cardiac muscle cells, pancreatic cells (e.g., islet cells), chondrocytes, osteocytes, skeletal muscle cells, smooth muscle cells, hepatocytes, hematopoietic cells, and adipocytes.
In a third aspect, the invention features a population of at least ten cells, wherein at least 30% of the cells are MNSCs substantially purified from a peripheral tissue of a postnatal mammal or progeny of the MNSCs, wherein the peripheral tissue includes a sensory receptor.
Preferably, at least 50% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs. More preferably, at least 75% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs. Most preferably, at least 90%, 95%, or even 100% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs. The MNSCs may be cultured for extended periods of time. Thus, the population of cells may have been in culture for at least thirty days, sixty days, ninety days, or longer (e.g., one year or more). Preferably, the population is at least twenty cells, and may be more than fifty cells, a thousand cells, or even a million cells or more.
In a fourth aspect, the invention features a pharmaceutical composition including (i) a mitotic or differentiated cell that is the progeny of a MNSC substantially purified from a peripheral tissue of a postnatal mammal, wherein the peripheral tissue includes a sensory receptor, and (ii) a pharmaceutically acceptable carrier, auxiliary or excipient.
In a fifth, related aspect, the invention features a pharmaceutical composition including (i) a MNSC substantially purified from a peripheral tissue of a postnatal mammal, wherein the peripheral tissue includes a sensory receptor, and (ii) a pharmaceutically acceptable carrier, auxiliary or excipient.
Preferably, the composition of the fourth or fifth aspect includes a population of cells, wherein at least 30%, 50%, 75%, 90%, 95%, or even 100% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs. The composition may include one or more types of cells selected from a group consisting of MNSCs, or neurons, oligodendrocytes, Schwann cells, and astrocytes which are progeny of MNSCs.
In a sixth aspect, the invention features a method of producing a population of at least ten cells, wherein at least 30% of the cells are MNSCs substantially purified from a peripheral tissue of a postnatal mammal or progeny of the MNSCs, wherein the peripheral tissue includes a sensory receptor, the method including: (a) providing the peripheral tissue from the mammal; (b) culturing the tissue under conditions in which MNSCs proliferate and in which at least 25% of the cells that are not MNSCs die; and (c) continuing culture step (b) until at least 30% of the cells are MNSCs or progeny of the MNSCs.
In a seventh aspect, the invention features another method of producing a population of at least ten cells, wherein at least 30% of the cells are MNSCs substantially purified from skin tissue of a postnatal mammal or progeny of the MNSCs, the method including: (a) providing the skin tissue from the mammal; (b) culturing the tissue under conditions in which MNSCs proliferate and in which at least 25% of the cells that are not MNSCs die; (c) separating the MNSCs from cells that are not MNSCs; and (d) repeating steps (b) and (c) until at least 30% of the cells are MNSCs or progeny of the MNSCs.
Suitable culture conditions for step (b) of the sixth and seventh aspects are preferably as follows: (i) triturating or otherwise mechanically separating tissue into single cells or cell clusters and placing into culture medium; (ii) culturing the cells in culture medium and under conditions (e.g., DMEM: Ham""s F-12 medium containing B-27 supplement, antibacterial and antifungal agents, 5-100 ng/ml bFGF, and 2-100 ng/ml EGF) that allows for the proliferation of MNSCs but does not promote, to the same extent, proliferation of cells that are not MNSCs; and (iii) culturing the mechanically separated tissue for three to ten days, during which time the MNSCs proliferate in suspension but non-MNSCs do not proliferate in suspension (these cells either attach to the plastic or they die). Preferably, at least 50% of the cells in suspension surviving after the period in culture are MNSCs or progeny of the MNSCs, more preferably, at least 75% of the cells are MNSCs or progeny of the MNSCs, and, most preferably, at least 90% or even 95% of the surviving cells are MNSCs or progeny of the MNSCs.
In an eighth aspect, the invention features a method of treating a patient having a disease associated with cell loss. The method includes the step of transplanting the multipotent neural stem cells of the invention into the region of the patient in which there is cell loss. Preferably, prior to the transplanting step, the method includes the steps of providing a culture of peripheral tissue containing sensory receptors from the patient and isolating a multipotent neural stem cell from the peripheral tissue. After transplanation, the method may further include the step of differentiating (or allowing the differentiation of) the MNSCs into a desired cell type to replace the cells that were lost. Preferably, the region is a region of the CNS or PNS, but can also be cardiac tissue, pancreatic tissue, or any other tissue in which cell transplantation therapy is possible.
In a ninth aspect, the invention features a kit including a MNSC substantially purified from a peripheral tissue of a postnatal mammal, or a mitotic or differentiated cell that is the progeny of the MNSC, wherein the peripheral tissue from which the MNSC is purified includes a sensory receptor. Preferably, the kit includes a population of cells, wherein at least 30%, 50%, 75%, 90%, or even 95% of the cells are MNSCs substantially purified from the peripheral tissue or progeny of the MNSCs.
In a tenth aspect, the invention features a kit for purifying MNSCs from peripheral tissue containing sensory receptors. The kit includes media or media components that allow for the substantial purification of MNSCs of the present invention. The kit may also include media or media components that allow for the differentiation of the MNSCs into the desired cell type(s). Preferably, the kit also includes instructions for its use.
In one preferred embodiment of each of the foregoing aspects of the invention, the peripheral tissue is skin tissue. In another preferred embodiment, the peripheral tissue is olfactory epithelium or tongue tissue. In still another embodiment, the peripheral tissue of the first aspect specifically excludes olfactory epithelium and tongue tissue.
The peripheral tissue can be from a newborn mammal, a juvenile mammal, or an adult mammal. Preferred mammals include, for example, humans, non-human primates, mice, pigs, and rats. The MNSCs can be derived from peripheral tissue of any individual, including one suffering from a disease or from an individual immunologically compatible to an individual suffering from a disease. In a preferred embodiment, the cells, or progeny of the cells, are transplanted into the CNS or PNS of an individual having a neurodegenerative disease and the individual is the same individual from whom the MNSCs were purified. Following transplantation, the cells can differentiate into cells that are lacking or non-functional in the disease.
Preferably, the MNSCs express nestin and/or glutamic acid decarboxylase. The MNSCs of the present invention can, under appropriate conditions, differentiate into neurons, astrocytes, Schwann cells, oligodendrocytes, and/or non-neural cells (e.g., cardiac cells, pancreatic cells, smooth muscle cells, adipocytes, hepatocytes, etc.).
MNSCs can be stably or transiently transformed with a heterologous gene (e.g., one encoding a therapeutic protein, such as a protein which enhances cell divisions or prevents apoptosis of the transformed cell or other cells in the patient, or a cell fate-determining protein).
By xe2x80x9cmultipotent neural stem cellxe2x80x9d or xe2x80x9cMNSCxe2x80x9d is meant a cell that (i) has the potential of differentiating into at least two cell types selected from a neuron, an astrocyte, and an oligodendrocyte, and (ii) exhibits self-renewal, meaning that at a cell division, at least one of the two daughter cells will also be a stem cell. The non-stem cell progeny of a single MNSC are capable of differentiating into neurons, astrocytes, Schwann cells, and oligodendrocytes. Hence, the neural stem cell is xe2x80x9cmultipotentxe2x80x9d because its progeny have multiple differentiative pathways. The MNSC may also have the potential to differentiate as another cell type (e.g., a skin cell, a hematopoietic cell, a smooth muscle cell, a cardiac muscle cell, a skeletal muscle cell, or a pancreatic cell).
By xe2x80x9csubstantially purifiedxe2x80x9d is meant that the desired cells (e.g., MNSCs) are enriched by at least 30%, more preferably by at least 50%, even more preferably by at least 75%, and most preferably by at least 90% or even 95%.
By xe2x80x9ctherapeutic proteinxe2x80x9d is meant a protein that improves or maintains the health of the cell expressing the protein or a that of a cell in proximity to the expressing cell. Example therapeutic proteins include, without limitation, growth factors (NGF, BDNF, NT-3, NT-4/5, HGF, TGF-xcex2 family members, PDGF, GDNF, FGF, EGF family members, IGF, insulin, BMPs, Wnts, hedgehogs, and heregulins) cytokines (LIF, CNTF, TNFM interleukins, and gamma-interferon), and anti-apoptotic proteins (LAP proteins, Bcl-2 proteins, Bcl-XL, Trk receptors, Akt, PI3 kinase, Gab, Mek, E1B55K, Raf, Ras, PKC, PLCxcex3, FRS2, rAPs/SH2B, and xcex94Np73).
By xe2x80x9cperipheral tissues containing sensory receptorsxe2x80x9d is meant a tissue that is not derived from neuroectoderm and specifically includes olfactory epithelium, tongue, skin (including dermis and epidermis), and mucosal layers of the body (e.g., mouth, reproductive system).
By a xe2x80x9cpopulation of cellsxe2x80x9d is meant a collection of at least ten cells. Preferably, the population consists of at least twenty cells, more preferably at least one hundred cells, and most preferably at least one thousand or even one million cells. Because of the MNSCs of the present invention exhibit a capacity for self-renewal, they can be expanded in culture to produce populations even billions of cells.
By xe2x80x9cpostnatalxe2x80x9d is meant an animal that has been born at term.
By xe2x80x9ca disease characterized by failure of a cell typexe2x80x9d is meant one in which the disease phenotype is the result of loss of cells of that cell type or the loss of function of cells of that cell type.
Other features and advantages of the present invention will become apparent from the following detailed description and the claims. It will be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of example only, and various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
We have substantially purified multipotent neural stem cells (MNSCs) from peripheral tissues of mammals, including skin, olfactory epithelium, and tongue. These cells proliferate in culture, so that large numbers of stem cells can be generated. These cells can be induced to differentiate, for example, into neurons, astrocytes, and/or oligodendrocytes by altering the culture conditions. They can also be induced to differentiate into non-neural cells such as smooth muscle cells, hematopoietic cells, adipocytes, hepatocytes, and the like. The substantially purified neural stem cells are thus useful for generating cells for use, for example, in autologous transplants for the treatment of degenerative disorders or trauma (e.g., spinal cord injury). In one example, MNSCs may be differentiated into dopaminergic neurons and implanted in the substantia nigra or striatum of a Parkinson""s disease patient. In a second example, the cells may be used to generate oligodendrocytes for use in autologous transplants for the treatment of multiple sclerosis. In still another example, the MNSCs may be used to generate Schwann cells for treatment of spinal cord injury, cardiac cells for the treatment of heart disease, or pancreatic islet cells for the treatment of diabetes If desired, in any of the foregoing examples, the cells may be genetically modified to express, for example, a growth factor, an anti-apoptotic protein, or another therapeutic protein.
The MNSCs display some similarities to stem cells derived from mammalian forebrain, but also possess some distinctive differences. In particular, when the MNSCs of the present invention differentiate in the presence of serum, about 5-20% of the differentiated cells express neuronal markers, whereas differentiated forebrain stem cells generate only a small percentage of neurons. Moreover, significant numbers of dopaminergic neurons are found in differentiated cultures of MNSCs of the present invention, whereas such neurons have not been observed in cultures of forebrain stem cells differentiated in serum.
The MNSCs of this invention may be used to prepare pharmaceutical compositions that can be administered to humans or animals for cell therapy. The cells may be undifferentiated or differentiated prior to administration. Dosages to be administered depend on patient needs, on the desired effect, and on the chosen route of administration.
The invention also features the use of the cells of this invention to introduce therapeutic compounds into the diseased, damaged, or physically abnormal CNS, PNS, or other tissue. The MNSCs thus act as a vector to transport the compound. In order to allow for expression if the therapeutic compound, suitable regulatory elements may be derived from a variety of sources, and may be readily selected by one with ordinary skill in the art. Examples of regulatory elements include a transcriptional promoter and enhancer or RNA polymerase binding sequence, and a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the vector employed, other genetic elements, such as selectable markers, may be incorporated into the recombinant molecule. The recombinant molecule may be introduced into the stem cells or the cells differentiated from the stem cells using in vitro delivery vehicles such as retroviral vectors, adenoviral vectors, DNA virus vectors and liposomes. They may also be introduced into such cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as incorporation of DNA into liposomes. The genetically altered cells may be encapsulated in microspheres and implanted into or in proximity to the diseased or damaged tissue.
Preferably, the MNSCs are used for the treatment of neurological disease, but may be used as a source of non-neural cells. PCT publication WO99/16863 describes the differentiation of forebrain MNSCs into cells of the hematopoietic cell lineage in vivo. The MNSCs of the present invention appear to be more plastic and thus are highly likely to also be capable of differentiating into non-neural cells types, such as hematopoietic cells. Accordingly, the invention features methods of treating a patient having any disease or disorder characterized by cell loss by administering MNSCs of the present invention (or cells derived from these cells) to that patient and allowing the cells to differentiate to replace the cells lost in the disease or disorder. For example, transplantation of MNSCs and their progeny provide an alternative to bone marrow and hematopoietic stem cell transplantation to treat blood-related disorders. Other uses of the MNSCs are described in Ourednik et al. (Clin. Genet. 56:267-278, 1999), hereby incorporated by reference.